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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
  • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
  • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

• the present invention relates to a novel protein, in particular mutant luciferase enzymes which show distinctive properties as compared to corresponding wild type enzyme, to DNA encoding these proteins, to the use of these enzyme in assays and to test kits containing them.
• Firefly luciferase catalyses the oxidation of luciferin in the presence of ATP, Mg 2+ and molecular oxygen with the resultant production of light. This reaction has a quantum yield of about 0.88.
• the light emitting property has led to its use in a wide variety of luminometric assays where ATP levels are being measured. Examples of such assays include those which are based upon the described in EP-B-680515 and WO 96/02665 but many others are used routinely in laboratories.
• Luciferase is obtainable directly from the bodies of insects, in particular beetles such as fireflies or glow-worms.
• Particular species from which luciferases have been obtained include the Japanese GENJI or KEIKE fireflies, Luciola cruciata and Luciola lateralis , the East European firefly Luciola mingrelica , the North American firefly Photinus pyralis and the glow-worm Lampyris noctiluca.
• the colour of the light emitted by these enzymes when used in assays in the laboratory are broadly similar. It would be helpful if the wavelength could be altered, either to be more easily read by the specific detector, or for use in systems where multiple reporters are required, for example to monitor different events within the same sample.
• One way of distinguishing reporter molecules is to utilise luciferase molecules which emit light at distinct wavelengths. This may be achieved by using reporter molecules comprising luciferases derived from different species of beetle or glow-worm.
• An alternative strategy however is to produce mutant luciferases using recombinant DNA technology, so as to produce a variation in the wavelength of the signal. Examples of such mutants are provided in WO 95/18853.
• the heat stability of wild and recombinant type luciferases is such that they lose activity quite rapidly when exposed to temperatures in excess of about 30° C., particularly over 35° C. This instability causes problems when the enzyme is used or stored at high ambient temperature, or if the assay is effected under high temperature reaction conditions, for example in order to increase reaction rate.
• Mutant luciferases having increased thermostability are known from EP-A-524448 and WO/95/25798. The first of these describes a mutant luciferase having a mutation at position 217 in the Japanese firefly luciferase, in particular by replacing a threonine residue with an isoleucine residue.
• mutant luciferases having over 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis but in which the amino acid residue corresponding to residue 354 of Photinus pyralis or 356 of the Luciola species is mutated such that it is other than glutamate, and in particular is other than glutamate, aspartate, proline or glycine.
• proteins which have luciferase activity and at least 60% similarity to wild-type luciferase such as those from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis enzyme, but which include mutations at various positions in the protein, including amongst others, (a) the amino acid residue corresponding to residue 214 in Photinus pyralis luciferase and to residue 216 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase; or
• the applicants have found that by mutating (or introducing) an amino acid at a different position within the luciferase protein, large shifts in the wavelength of the emitted light may be achieved and/or the enzyme has improved thermostability. Furthermore, the proton flux of emitted light may be improved, making the enzyme better suited to in vivo assays where glow kinetics are precluded or in vitro assays where CoA or other ‘glow kinetic inducing’ compounds are not present.
• the present invention provides a recombinant protein having luciferase activity and at least 60% similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase.
• Wild-type luciferase sequences which can form the basis of the recombinant forms of the invention include Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria paroula, Pyrophorus plagiophthalamus Lampyris noctiluca, Pyrocoelia nayako, Photinus pennsylvanica or Phrixothrix (railroad-worms—see Biochem. 38 (1999) 8271-8279).
• Bioluminescent enzymes from species that can use the substrate D-luciferin (4,5-dihydro-2-[6-hydroxy-2-benzothiazolyl]-4-thiazole carboxylic acid) to produce light emission may form the basis of the mutant enzymes of the invention.
• Particular wild-type luciferase sequences which can form the basis of the recombinant forms of the invention include Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis, Hotaria paroula, Pyrophorus plagiophthalamus Lampyris noctiluca, Pyrocoelia nayako and Photinus pennsylvanica.
• amino acid residue corresponding to residue 357 in Photinus pyralis luciferase most wild-type sequences have an acid residue (aspartic acid or glutamic acid) at this position.
• the exception to this is some forms of the luciferase of Photinus pennsylvanica in which the corresponding residue (355) is the non-polar residue, valine or some forms of Phrixothrix luciferase where the corresponding position is V354 in Pv GR or in Ph RE , where it is L354 leucine.
• the amino acid used as a substitute amino acid at this position is other than aspartic acid, glutamic acid, valine or leucine.
• an acidic amino acid residue is replaced with a non-acidic residue, including basic amino acids such as lysine or arginine, non-polar amino acids such as leucine, valine or isoleucine, uncharged polar amino acids such as tyrosine, asparagine, glutamine, phenylalanine, serine, tryptophan or threonine.
• a non-acidic residue including basic amino acids such as lysine or arginine, non-polar amino acids such as leucine, valine or isoleucine, uncharged polar amino acids such as tyrosine, asparagine, glutamine, phenylalanine, serine, tryptophan or threonine.
• it may be replaced with an uncharged polar amino acid such as tyrosine, asparagine, serine or threonine.
• Particularly preferred amino acid residues for substitution at this position are tyrosine, phenylalanine or trypto
• wild-type sequences include non-acidic amino acid residues at this position, they are suitably mutated into different non-acidic residues.
• D357Y mutant Photinus pyralis luciferase emits light at a wavelength of some 612 nm as compared to the wild-type enzyme which emits light at a wavelength of 562 nm.
• a wavelength shift of 50 nm has considerable potential for use in assay applications as a shift of this magnitude can be readily defined spectrally.
• Different coloured luciferases could be employed as reporter molecules in gene expression studies, enabling the simultaneous monitoring of more than one gene, for example as described in WO 95/18853. Multiple analyte testing could also be performed with luciferase as labels.
• red in colour is particularly useful in assay methodology.
• a red mutant could be useful when analysing a solution for ATP which contained pigments or other compounds which may absorb shorter wavelengths of light.
• a red coloured solution would not absorb red light.
• red coloured solutions which are frequently the subject of such analysis include blood samples or a solution of eukaryotic cell culture medium which may contain a red coloured pH indicator.
• a photomultiplier tube used in photocathode spectral analysis can be set to detect either one or both peaks generated in a single sample. In other words, it is possible to distinguish between photon flux from a red and green emitter in the same sample.
• an assay for determining the presence in a sample of CoA comprises adding to a sample suspected of containing CoA, luciferase as described above together with other reagents which are required to bring about a luciferase/luciferin reaction, measuring the wavelength of light emitted from the sample and relating this to the presence or absence of CoA.
• Such an assay may be useful in the detection of the state of growth or activity of cells, for example microorganisms or eukaryotic cells.
• the concentration of CoA in E. coli cells is relatively high, and varies considerably with metabolic status.
• the mutant enzymes of the invention can be used to monitor the metabolic status of an organism, particularly the in vivo concentration of the CoA, since the wavelength of the emission varies depending upon the CoA concentration.
• Such assays may be particularly useful in situations where CoA is an important primary metabolite in the production of antibiotics (e.g. in streptomycetes).
• Cellular CoA concentrations are also an important indicator of fatty acid biosynthesis and vary with the starvation status of the cell.
• a number of metabolic disorders such as carcinogenesis and diabetes, show abnormalities in the fatty acid metabolites and consequently unusual CoA levels.
• Assays of the invention may be used in the diagnosis of such conditions.
• the CoA levels from within a cell sample may be determined by measuring the wavelength of light emitted from a luciferase of the invention, used in the assay. This result may be compared with that obtained from a sample of healthy cells to determine whether the wavelength has changed and thus that a modified CoA level is present. This may be indicative of a disease state in the patient.
• Cells are suitably lysed prior to assay using a known lytic agent.
• the bridging residues may be flexible enough to disengage when the enzyme is in solution to provide a larger pocket ( ⁇ 12 ⁇ deep and ⁇ 8 ⁇ wide) which allows CoA binding. This is consistent with the energy calculations.
• the proteins may contain further mutations in the sequence provided the luciferase activity of the protein is not unduly compromised.
• the mutations suitably enhance the properties of the enzyme or better suit it for the intended purpose in some way. This may mean that they result in enhanced thermostability and/or colour shift properties, and/or the K m for ATP of the enzymes. Examples of mutations which give rise to colour shifts are described in WO95/18853. Mutations which affect K m values are described for example in WO 96/22376 and International Patent Application No. PCT/GB98/01026.
• mutant luciferases of the invention may include other specific mutations which enhance thermostability as compared to wild-type luciferase.
• amino acid residue corresponding to position 215 in Photinus pyralis luciferase or (217 in Luciola luciferase) is a different hydrophobic amino acid
• (k) amino acid residue corresponding to amino acid residue 310 of the Photinus pyralis luciferase or to residue 312 of Luciola mingrelica, Luciola cruciata or Luciola lateralis ; is different to the amino acid which appears in the corresponding wild type sequence and wherein the luciferase enzyme has increased thermostability as compared to an enzyme having the amino acid of the corresponding wild-type luciferase at this position.
• proteins of the invention are mutated wild-type luciferases where more than one amino acid, for example up to 100 amino acid residues, preferably no more than 40 amino acids, and more preferably up to 30 amino acids, are different to the amino acid at the corresponding position in the appropriate wild-type enzyme.
• the protein of the invention comprises luciferase of Photinus pyralis , wherein, in addition to the mutation at the 357 position as described above, at least one of;
• amino acid residue corresponding to amino acid 354 of the Photinus pyralis luciferase is other than glutamate;
• amino acid residue corresponding to residue 295 in Photinus pyralis luciferase is other than phenylalanine;
• amino acid residue corresponding to amino acid 14 of the Photinus pyralis luciferase is other than phenylalanine
• amino acid residue corresponding to amino acid 35 of the Photinus pyralis luciferase is other than leucine
• amino acid residue corresponding to amino acid residue 105 of the Photinus pyralis luciferase is other than alanine
• amino acid residue corresponding to amino acid residue 234 of the Photinus pyralis luciferase is other than aspartic acid
• amino acid residue corresponding to amino acid residue 420 of the Photinus pyralis luciferase is other than serine;
• amino acid residue corresponding to amino acid residue 310 of the Photinus pyralis luciferase is other than histidine.
• the protein of the invention comprises protein the luciferase sequence of Luciola mingrelica, Luciola cruciata or Luciola lateralis enzyme, and wherein, in addition to the mutation at position 359 as described above, at least one of
• amino acid residue corresponding to amino acid 356 of the Photinus pyralis luciferase is other than glutamate;
• thermostability The particular substituted amino acids in any case which give rise to enhanced thermostability can be determined by routine methods as illustrated hereinafter. In each case, different substitutions may result in enhanced thermostability. Substitution may be effected by site-directed mutagenesis of DNA encoding native or suitable mutant proteins as would be understood by the skilled person. The invention in this case is associated with the identification of the positions which are associated with thermostability.
• hydrophilic amino acid residues may, in some cases be preferably substituted with hydrophobic amino acid residues and vice versa.
• acidic amino acid residues may be substituted with basic residues.
• the protein may comprise a protein having luciferase activity and at least 60% similarity to luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis enzyme wherein in the sequence of the enzyme, at least one of;
• the polar amino acid threonine is suitably replaced with a non polar amino acid such as alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine.
• a particularly preferred substitution for the threonine residue corresponding to residue 214 in Photinus pyralis is alanine.
• a more preferred substitution is cysteine.
• different polar residues such as asparagine at this position may also enhance the thermostability of the corresponding enzyme having threonine at this position.
• luciferase enzymes include glycine ( Luciola mingrelica, Hotaria paroula ), asparagine ( Pyrophorus plagiophthalamus , GR, YG, YE and OR, Luciola cruciata, Luciola lateralis, Lampyris noctiluca, Pyrocelia nayako Photinus pennsylvanica LY, KW, J19) and serine ( Phrixothix ). These may advantageously be substituted with non-polar or different non-polar side chains such as alanine and cysteine.
• the nonpolar amino acid isoleucine is suitably replaced with a different non polar amino acid such as alanine, glycine, valine, lecine, proline, phenylalanine, methionine, tryptophan or cysteine.
• alanine glycine
• valine valine
• lecine proline
• phenylalanine methionine
• tryptophan cysteine.
• Other amino acids appearing at this position in wild type sequences include serine and asparagine.
• these polar residues are substituted by non-polar residues such as those outlined above.
• a particularly preferred substitution for the residue corresponding to residue 232 in Photinus pyralis luciferase and to residue 234 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase at group is alanine.
• the non-polar amino acid phenylalanine is suitably replaced with a different non polar amino acid, such as alanine, leucine, glycine, valine, isoleucine, proline, methionine, tryptophan or cysteine.
• a particularly preferred substitution for the phenylalanine residue corresponding to residue 214 in Photinus pyralis luciferase is leucine.
• Mutation at the amino acid residue corresponding to amino acid 14 of the Photinus pyralis luciferase or to amino acid 16 in Luciola luciferase (13 in Phrixothrix luciferase ) is also possible.
• This amino acid residue (which is usually phenylalanine, but may also be leucine, serine, arginine or in some instances tyrosine) is suitably changed to a different amino acid, in particular to a different nonpolar amino acid such as alanine, valine, leucine, isoleucine, proline, methionine or tryptophan, preferably alanine.
• Mutation at the amino acid residue corresponding to amino acid 35 of the Photinus pyralis luciferase or to amino acid residue 37 in Luciola mingrelica luciferase (38 in other Luciola spp.) may also be effective.
• This amino acid varies amongst wild type enzymes, which may include leucine ( Photinus pyralis ) but also lysine, histidine, glycine, alanine, glutamine and aspartic acid at this position.
• amino residue at this position is substituted with a non-polar amino acid residue or a different non-polar amino acid such as alanine, valine, phenylalanine, isoleucine, proline, methionine or tryptophan.
• a preferred amino acid at this position is alanine, where this is different to the wild-type enzyme.
• Mutations at the amino acid corresponding to position 14 of the Photinus pyralis sequence and/or mutation at the amino acid residue corresponding to amino acid 35 of the Photinus pyralis luciferase are preferably not the only mutation in the enzyme.
• Changes of the amino acid residue corresponding to residue 105 in Photinus pyralis luciferase and to residue 106 of Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferase, (102 in Phrixothrix ) may also affect the thermostability of the protein.
• the amino acid at this position is a non-polar amino acid alanine or glycine, or serine in Phrixothrix . These are suitably changed for different non-polar amino acids.
• the non-polar amino acid alanine is suitably replaced with a different non polar amino acid, such as phenylalanine, leucine, glycine, valine, isoleucine, proline, methionine or tryptophan.
• a particularly preferred substitution for the alanine residue corresponding to residue 105 in Photinus pyralis luciferase is valine.
• the amino acid at this position is aspartic acid or glycine and in some cases, glutamine or threonine. These are suitably changed for non-polar or different non-polar amino acids as appropriate.
• the amino acid residue is aspartic acid is suitably replaced with a non polar amino acid, such as alanine, leucine, glycine, valine, isoleucine, proline, methionine or tryptophan.
• a particularly preferred substitution for the phenylalanine residue corresponding to residue 234 in Photinus pyralis luciferase is glycine.
• a non-polar amino acid residue such as glycine is present at this position (for example in Luciola luciferase )
• this may be substituted with a different non-polar amino acid.
• the amino acid at this position is an uncharged polar amino acid serine or threonine or glycine. These are suitably changed for different uncharged polar amino acids.
• the serine may be replaced with asparagine, glutamine, threonine or tyrosine, and in particular threonine.
• the amino acid residue at this position varies amongst the known luciferase proteins, being histidine in Photinus pyralis, Pyrocelia nayako, Lampyris noctiluca and some forms of Photinus pennsylanvanica luciferase, threonine in Luciola mingrelica, Hotaria paroula and Phrixothix (where it is amino acid 307) luciferase, valine in Luciola cruciata and Luciola lateralis , and asparagine in some Pyrophorus plagiophthalamus luciferase.
• the amino acid at this position is hydrophilic amino acid which may be changed for a different amino acid residue which increases thermostability of the enzyme.
• a particularly preferred substitution for the histidine residue corresponding to residue 310 in Photinus pyralis luciferase is arginine.
• the protein also has the amino acid at position corresponding to amino acid 354 of the Photinus pyralis luciferase (356 in Luciola luciferase ) changed from glutamate, in particular to an amino acid other than glycine, proline or aspartic acid.
• the amino acid at this position is tryptophan, valine, leucine, isoleucine are asparagine, but most preferably is lysine or arginine.
• This mutation is described in WO 95/25798. It has been found that hydrophobic residues at this position enhance the wavelength shift of the enzyme, Furthermore, the presence of a large hydrophobic (V or I), polar (N) or positively charged (K or R) amino acid at position 354 enhances thermostability.
• the protein also has the amino acid at the position corresponding to amino acid 217 in Luciola luciferase (215 in Photinus pyralis ) changed to a hydrophobic amino acid in particular to isoleucine, leucine or valine as described in EP-A-052448.
• Proteins of the invention include both wild-type and recombinant luciferase enzymes. They have at least 60% similarity to wild sequences such as those of Photinus pyralis, Luciola mingrelica, Luciola cruciata or Luciola lateralis enzyme in the sense that at least 60% of the amino acids present in the wild-type enzymes are present in the proteins of the invention. Such proteins can have a greater degree of similarity, in particular at least 70%, more preferably at least 80% and most preferably at least 90% to the wild-type enzymes listed above. Similar proteins are of this type include allelic variants, proteins from other insect species as well as recombinantly produced enzymes.
• low stringency conditions can be defined as 3 ⁇ SCC at about ambient temperature to about 65° C.
• high stringency conditions as 0.1 ⁇ SSC at about 65° C.
• SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3 ⁇ SSC is three times as strong as SSC and so on.
• the similarity of a particular sequence to the sequences of the invention may be assessed using the multiple alignment method described by Lipman and Pearson, (Lipman, D. J. & Pearson, W. R. (1985) Rapid and Sensitive Protein Similarity Searches, Science, vol 227, pp 1435-1441).
• the sequences for which similarity is to be assessed should be used as the “test sequence” which means that the base sequence for the comparison, such as the sequence of Photinus pyralis or any of the other sequences as recorded in Ye et al., supra., should be entered first into the algorithm.
• proteins of the invention are wild-type luciferase sequence with one or more of the mutations as outlined above.
• the invention further provides nucleic acids which encode the luciferases as described above.
• the nucleic acids are based upon wild-type sequences which are well known in the art. Suitable mutation to effect the desired mutation in the amino acid sequence would be readily apparent, based upon a knowledge of the genetic code.
• the nucleic acid is a synthetic gene.
• the synthetic gene is engineered to remove codons rarely found in highly expressed genes from common expression hosts such as E. coli and, at the same time, avoid the introduction of codons rarely found in genes coding for beetle luciferases. This approach ensures that the new gene has a codon utilisation that is optimal for both E. coli and insect expression systems.
• the codons for the amino acids arg, leu, ile, gly and pro were changed to CGT or CGC (arg), CTG, CTT or CTC (leu), ATC or ATT (ile), GGT or GGC (gly), and CCG CCA or CCT (pro), thus eliminating rare codons.
• SEQ ID NO 1 the synthetic gene illustrated below
• FIG. 14 this resulted in a total of 139 silent mutations creating 62 new non-rare codons (11% of the total).
• the first 8 nucleotides shown in FIG. 14 form part of the ribosome binding site and thus do not code.
• the coding sequence begins with the methionine residue indicated by an up arrow. This coding sequence and closely similar sequences, for example sequences which have at least 90% similarity or preferably at least 95% similarity form a preferred aspect of the invention.
• Another useful feature which may be employed when producing a synthetic assembly is the incorporation of new unique restriction sites. These sites make mutagenesis, in particular combinatorial cassette mutagenesis, of the gene simpler and more efficient. In particular, it may be desirable to create unique restriction sites within the cDNA coding for subdomain B in the enzyme. Additionally creation of a unique restriction site at the extreme 3′ end of the gene to allow simple fusions and/or removal of the peroxisome targeting sequence may be advantageous.
• thermostabilising amino acid changes T214C, E354K and D357F were engineered to introduce the thermostabilising amino acid changes T214C, E354K and D357F into the polypeptide.
• the nucleic acids of the invention are suitably incorporated into an expression vector such as a plasmid under the control of control elements such as promoters, enhancers, terminators etc.
• a host cell for example a prokaryotic or eukaryotic cell such as a plant or animal cell, but in particular a prokaryotic cell such as E. coli so that the cell expresses the desired luciferase enzyme.
• Culture of the thus transformed cells using conditions which are well known in the art will result in the production of the luciferase enzyme which can then be separated from the culture medium.
• the cells are plant or animal cells, plants or animals may be propagated from said cells.
• the protein may then be extracted from the plants, or in the case of transgenic animals, the proteins may be recovered from milk.
• Vectors, transformed cells, transgenic plants and animals and methods of producing enzyme by culturing these cells all form further aspects of the invention.
• the Photinus pyralis D357Y mutant luciferase was created by random mutagenesis as described hereinafter. It was found that the D357Y single point mutation produces a large colour shift in the wavelength of light emitted and also has greater thermostability than wild type luciferase. Further investigations have revealed that a range of substitutions at this position give rise to good thermostability and/or to large colour shifts.
• mutant enzymes of Photinus pyralis which fall within the scope of the invention include the following:
• the mutations for the creation of the above mutants were introduced to the luciferase gene on plasmid pET23 by site-directed mutagenesis, (PCR) or combinatorial cassette mutagenesis.
• PCR site-directed mutagenesis
• the oligonucleotides added to the PCR reaction in order to effect the relevant mutations are given below.
• Luciferase proteins of the invention will advantageously be employed in any bioluminescent assay which utilises the luciferase/luciferin reaction as a signalling means. There are many such assays known in the literature. The proteins may therefore be included in kits prepared with a view to performing such assays, optionally with luciferin and any other reagents required to perform the particular assay.
• FIG. 1 is a log graph showing percent % remaining activity versus time of 45° C. incubation of several mutant enzymes in accordance with the invention
• FIG. 2 shows the spectral peaks obtained by incubating E. coli cells expressing luciferase enzymes in a citrate buffer with D-luciferin where the enzyme used is (a) recombinant wild-type Photinus pyralis luciferase, (b) a D357K mutant, (c) a D357N mutant, (d) a D357W mutant, (e) a D357I mutant, (f) a D357F mutant, (g) a D357Y mutant and (h) a double mutant E354I+D357Y;
• FIG. 3 is a graph showing the % remaining activity versus time of three mutant enzymes, E354I, D357Y and the double mutant (DM) E354I/D357Y;
• FIG. 4 shows the emission spectra of (a) recombinant wild type enzyme and (b) the double mutant (DM) E354I/D357Y;
• FIG. 5 is a graph showing the rate decay of photon emissions of recombinant wild-type ( ⁇ ) r-wt and a D357K mutant enzyme (:)
• FIG. 6 shows molecular modelling diagram, illustrating a potential CoA binding pocket within the luciferase enzyme
• FIG. 7 shows the in vivo bioluminescent spectra emitted by E. coli cells expressing mutant P. pyralis luciferase D357Y (a) growth on LB; (b) growth on minimal medium and sodium acetate; (c) growth on minimal medium and glucose;
• FIG. 8 shows the in vivo bioluminescent spectra emitted by E. coli cells expressing mutant P. pyralis luciferase E354K/D357M (a) growth on LB; (b) growth on minimal medium and sodium acetate; (c) growth on minimal medium and glucose;
• FIG. 9 is a graph showing the effect of CoA on spectral distribution of light emitted by mutant P. pyralis luciferase D357Y;
• FIG. 10 is a graph showing the normalised data of the effect of CoA on spectral distribution of light emitted by mutant P. pyralis luciferase D357Y;
• FIG. 11 is a graph showing the effect of CoA on spectral distribution of light emitted by mutant P. pyralis luciferase E354I/D357Y ( FIG. 11 a ) and normalised data ( FIG. 11 b );
• FIG. 12 illustrates the restriction sites modifications utilised in the construction of a synthetic luciferase gene
• FIG. 13 illustrates constructs used in the synthesis of a luciferase gene
• FIG. 14 shows the cDNA sequence (SEQ ID NO 1) of the synthetic luciferase gene (including nucleotides 1-8 which form part of the ribosome binding site but are not coding) and the encoded amino acid sequence which starts at the methionine residue indicated by the up arrow (SEQ ID NO 2); and
• FIG. 15 illustrates the thermostability of mutants including the mutant encoded by the synthetic gene at 50° C.
• Two libraries of firefly ( Photinus pyralis ) luciferase created used error-prone PCR [M. Fromant et al., Anal. Biochem. (1995) 224, 347-353], were prepared.
• One library comprised of error-prone PCR products of the full length luc gene, cloned into the T7 expression system pET23a, (Novagen Inc., Madison, Wis., U.S.A.).
• a second library consisted of the error-prone PCR products of a short section of the luc gene, covering amino acids 199-352, cloned in the vector pBSK(+), (Stratagene, La Jolla, Calif., U.S.A.).
• the pET23a library was expressed in E. coli strain BL21(DE3), ( E. coli B F dcm ompT hsdS(r s ⁇ m s ⁇ ) gala (DE3)).
• the pBSK(+) library was expressed in HB101 E. coli cells, (supE44 ara14 galK2 lacY1 ⁇ (gpt-proA) 62 rpsL20 (Str r ) xyl-5 mtl-1 recA13 ⁇ (mrcC-nmr) HsdS ⁇ (r ⁇ m ⁇ ).
• pET23a and pBSK(+) both carry the gene for ⁇ -lactamase and confer ampicillin resistance to E. coli cells harbouring the plasmid.
• An E. coli strain was transformed with the prepared library by electroporation, using a BIORAD E. coli Pulser, and grown overnight at 37° C. on LB agar, containing ampicillin at a concentration of 50 ⁇ g/ml.
• the cells were transferred to nylon membranes, (Osmonics, Minnetonka, Minn., U.S.A.), and sprayed with luciferin solution (500 ⁇ m D-luciferin, potassium salt, in 100 mM sodium citrate buffer, pH 5.0).
• the colonies were viewed using an ALPHAIMAGERTM 1200 documentation and analysis system (Flowgen, Lichfield, Staffordshire, UK). This integrated the bioluminescence emitted over a specified period of time to produce an image of the light emitted by the colonies. The brightness of luminescence was taken as an indication of the thermostability of luciferase.
• thermostability Colonies were selected on the basis of brightness of light emitted and were isolated for further characterisation. In some screens, the E. coli colonies were incubated at 42° C. for 2 hours prior to screening so that the thermostable mutants could be selected. Colonies isolated from the primary screen were patched onto nylon membranes and also grown overnight in LB medium containing ampicillin. The patches were sprayed with luciferin solution and viewed in the ALPHAIMAGERTM. This secondary screen helped to positively identify clones for in vitro analysis of luciferase activity. E. coli clones expressing possible thermostable enzymes were assayed in vitro for luciferase activity and thermostability.
• the luciferase reaction was initiated by the addition of 10 ⁇ l crude cell extract to 100 ⁇ l Promega Luciferase Assay Cocktail (1 in 2 dilution). The resultant bioluminescence was measured using a Biotrace M3 luminometer.
• Crude cell extracts were prepared as described in the Promega technical bulletin no. 101. Aliquots of E. coli overnight cultures were lysed in cell culture lysis reagent, (25 mM Tris-phosphate pH7.8, 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexame-N,N,N′,N′-tetraacetic acid, 10% glycerol, 1% Triton X-100, 1.25 mg/ml hen lysozyme) for 10 minutes at room temperature. Crude lysate were then stored on ice prior to assay.
• DTT dithiothreitol
• Triton X-100 1.25 mg/ml hen lysozyme
• T 1/2 is the time taken for the enzyme to lose 50% of its original activity after incubation at a given temperature. T 1/2 values, (time for activity to reduce to 50% of original activity), were determined in crude extracts at 37° C. from log graphs of % remaining activity versus time (not shown).
• Plasmid DNA was prepared using the QIAGEN QIAprep Spin Miniprep Kit, (QIAGEN Ltd, Crawley, W.shire, UK), following the protocol for using a microcentrifuge (QIAprep Miniprep Handbook 04/98).
• D357Y is a particularly thermostable mutant, being the most thermostable luciferase, with a single amino acid change.
• site-directed mutagenesis was performed using the Stratagene QUIKCHANGETM Site-Directed Mutagenesis Kit, (Stratagene, La Jolla, Calif., U.S.A.).
• the plasmid pPW601a J54, (PJW, MOD report, 3/96) was used in all site-directed mutagenesis. All products of the mutagenesis reactions were transformed into E.
• Oligonucleotide primers were synthesized by Sigma-Genosys Ltd., Cambridge, UK and were designed using an intelligent doping system (A. R. Arkin et al., Bio-technology, (1992) 10, 297-300, W., Huang et al., Anal. Biochem. 218, 454-457] were used to design degenerate oligonucleotide primers to produce groups of possible mutations rather than using individual primers for each amino acid substitution.
• N is the number of colonies to be screened
• n is the number of possible codons at the target position
• the mutants obtained from site-directed mutagenesis were assayed for luciferase activity and characterised in time-dependent thermoinactivation studies.
• Mutants identified as desirable in this way were grown in 400 ml LB medium, containing ampicillin, to A 260 ⁇ 0.5. Luciferase expression was then induced by addition of isopropyl ⁇ -thiogalactoside (IPTG) to a final concentration of 1 mM. The cells were then incubated at 30° C., with shaking, for 3 hours prior to harvesting by centrifugation. The resultant cell pellet was resuspended in 10 ml B-PERTM Protein Extraction Reagent, (Pierce Chemical Company, Rochford, U.S.A.), 1 mM DTT to produce a crude extract, following the B-PERTM protocol for Maxi-Scale Bacterial Protein Extraction.
• Reconstituted Sigma Protease Inhibitor Cocktail 500 ⁇ l, (Product No. P8465, Sigma, Saint Louis, Mo., U.S.A.), was added to the BPERTM solution to inhibit endogenous proteases. The cell lysate was then centrifuged at 30 000 g for 30 minutes.
• the supernatant of the crude extract was subjected to fractionation with ammonium sulphate.
• the fraction that precipitated between 30% and 55% saturation contained luciferase activity.
• This material was resuspended in 0.5 ml Tris HCl pH8.0, 1 mM DTT and used for thermoinactivation and spectral studies.
• thermoinactivation buffer 50 mM potassium phosphate buffer pH7.8 containing 10% saturated ammonium sulphate, 1 mM dithiothreitol and 0.2% BSA.
• the in vivo luminescence of the mutants was assessed by eye in a dark room.
• the D357 mutants displayed a variety of colours in their luminescence spectra.
• D357Y, F and I showed significant shifts to longer wavelengths of emitted light.
• the change in colour of light emission appeared to be due, not only to a shift in ⁇ max , but to a difference in contributions to the spectra from different wavelengths of visible light.
• Recombinant wild type (r-wt) enzyme was used for comparison of ⁇ max of in vivo light emission of some of the 357 mutants.
• D357Y, F and I display considerable shifts in their wavelength maxima.
• D357Y was partially purified by ammonium sulphate precipitation, as described in the Example 1. This partially purified D357Y enzyme (5 ⁇ l) was mixed with 150 ⁇ l Promega Luciferase Assay Reagent. Another aliquot was mixed with an equivalent assay buffer in which CoA is absent, (25 mM Tris Tricine pH7.8, 5.0 mM MgSO 4 , 0.1 mM EDTA, 2 mM DTT, 470 ⁇ M D-luciferin, 530 ⁇ M ATP). The emission spectra of the two reactions were measured and are shown in FIGS. 9 and 10 .
• the double mutant displayed a marked increase in thermostability in comparison to the single mutants E354I and D357Y individually, (see FIG. 3 ).
• Thermoinactivation studies of partially purified double mutant confirmed the increased thermostability of the mutant, giving a t 1/2 value of 7.7 min when inactivated at 45° C.
• the double mutant displays a much deeper red colour of luminescence than the individual mutants of E354I and D357Y, displaying additivity of colour of luminescence.
• Emission spectra measured in vivo give a ⁇ max of 611 nm. However, the spectrum has a greater contribution of luminescence from the red region of wavelengths, leading to its deeper red appearance when visualised by eye.
• Emission spectra in crude extracts displayed a definite change in spectral shape and a wavelength shift of 44 nm, relative to rWT, (see FIG. 4 ).
• the in vivo emission spectrum of the double mutant shows both a sharpening of the bandwidth for the peak wavelength of emitted light (613 nm) and a decrease in the contribution from wavelengths of light in the region 540-560 nm.
• the in vivo bioluminescence of E. coli cells expressing the mutant D357K was observed to be very bright relative to the other mutants at this position.
• the flash kinetics of this enzyme was analysed using a luminometer, which could measure the rate of photon emission over time. Aliquots of E. coli cell free extracts containing recombinant wild type enzyme or the mutant D357 were added to a luciferase assay cocktail, which did not contain any reagents that would promote glow kinetics, e.g. coenzymeA.
• the rate of decay of photon emission measured over time (15s) for both enzymes was observed to be significantly slower for the mutant D357K ( FIG. 5 ).
• the mutant enzyme has reaction kinetics, which are inhibited to a lesser degree; over at least the first 15 seconds of the reaction, than the recombinant wild type enzyme.
• Two new unique restriction sites were introduced into the luc gene, in the plasmid pPW601a/J54, using two pairs of synthetic oligonucleotides (see below).
• a total of six silent mutations introduced a SpeI and a KpnI restriction site, 63 base pairs apart, within the gene. Plasmid containing these new sites was called pPW601aJ54SpeI/KpnI.
• the presence and proximity of these restriction sites makes it possible to use combinatorial cassette mutagenesis to explore the effects of random substitutions at amino acid positions 354 and 357 in the primary sequence of firefly luciferase.
• a pair of synthetic oligonucleotides was synthesised which when annealed created a double stranded cassette which could be ligated directly into plasmid pPW601aJ54SpeI/KpnI digested at the new restriction sites.
• the cassette was designed to introduce all possible combinations of the 20 naturally occurring amino acids at positions 354 and 357 in the primary sequence.
• Looplib2A 5′-ctagtgctattctgattacacccNNG/CggggatNNG/Caaaccgggc gcggtcggtaaagtggta-3′(SEQ ID NO 12)
• Looplib2B 5′-cactttaccgaccgcgcccggtttG/CNNatccccG/CNNgggtgtaa tcagaatagca-3′ (SEQ ID NO 13)
• each of the loop library oligonucleotides was mixed in a buffer containing 50 mM Tris-HCl pH 7.4, 25 mM NaCl, and heated to 100° C. for 3 min. This solution was then cooled slowly in a heating block to ⁇ 50° C. to anneal the complimentary sequences. The annealed oligonucleotides were then ligated into plasmid pPW601aJ54SpeI/KpnI, which had been digested with SpeI and KpnI. Aliquots of the ligation reaction were then used to transform E. coli HB101 cells using electroporation.
• Plasmid DNA was prepared from the 25 clones selected by in vivo screening and sequenced using gene specific sequencing primers. Mutations resulting in amino acid changes at positions 354 and 357 in the primary sequence were identified. One mutant, also contained an additional mutation resulting in an amino acid substitution at position I351 (Table 5).
• mutant luciferases were selected from the in vivo assays for thermostability. The majority of these luciferases also show large changes in the in vivo spectrum of emitted light with many showing greater contributions from longer wavelengths of light (>580 nm). A number of spectra also showed a significant narrowing of bandwidth around a single peak of 610-614 nm.
• E354 and D357 with a hydrophobic and an aromatic amino acid respectively e.g. E354V, D357Y results in the largest change in the in vivo spectrum which shows a single peak, of narrow bandwidth, around 612 nm.
• Cell free extracts of the selected clones were prepared by lysis and the thermostability of the luciferase from each extract was determined in a thermal inactivation experiment. 50 ⁇ l of each extract was placed in an eppendorf tube and incubated in a waterbath heated to 45° C. for 4, 9 and 16 minutes. At the appropriate timepoint the aliquot was removed and the remaining luciferase activity measured. Table 6 shows the percent remaining activity versus time for all mutant enzymes as well as recombinant wild type.
• thermostable luciferases were those with an aromatic amino acid at position 357 (Y, F or W) and a large hydrophobic (V or I), polar (N) or positively charged (K or R) amino acid at position 354.
• a 50 ml culture of cells was grown to mid log phase on LB medium and then harvested by centrifugation.
• the cell pellet was resuspended in 1 ml of sterile distilled water and a 100 ul aliquot of this suspension was then used to inoculate 5 ml of fresh LB, M9 minimal medium+2 mM sodium acetate or M9 minimal medium+2 mM glucose in a 25 ml Sterilin tube.
• the cultures were allowed to continue growing, at 37° C.
• Recombinant wild type Photinus pyralis enzyme and the mutant luciferases D357Y and E354I+D357Y were purified to homogeneity in order to analyse the effect of the cofactor coenzyme A on the spectrum of the bioluminescent reaction.
• All three luciferases were purified as fusions to a 143 amino acid carbohydrate binding module (CBM) from the anaerobic fungus Piromyces equii .
• CBM carbohydrate binding module
• This CBM has been shown to bind selectively to acid swollen cellulose and the soluble carbohydrates galactomannan and glucomannan, forming the basis for a simple single step affinity purification scheme.
• Luciferases fused to the CBM can be bound to cellulose in crude cell free extracts, washed, and then eluted selectively using soluble polysaccharides. Fusion proteins purified this way were used in assays to measure the wavelengths of emitted light in reactions containing different amounts of coenzyme A.
• Enzyme (5 ⁇ l) was added to 100 ⁇ l of assay reagent, 25 mM Tris-Tricine pH 7.8, 5.0 mM MgSO4, 0.1 mM EDTA, 530 ⁇ M ATP and 470 ⁇ M D-luciferin, containing different amounts of coenzyme A.
• FIGS. 9-11 show the effect of increasing concentrations of coenzyme A on the spectrum of light emitted by the purified luciferases D357 and E354I+D357Y.
• a synthetic luc gene was designed and assembled from oligonucleotide pairs using the synthesis strategy outlined above.
• the gene sequence was engineered to create a luciferase with the amino acids 214C, 354K and 357F.
• oligonucleotides Twenty-nine pairs of overlapping synthetic oligonucleotides were synthesised by Sigma-Genosys Ltd, purified by PAGE and ligated in three assemblies of approximately 550 bp (IDRIS 1, 2 & 3, FIG. 13 ). Each assembly was then ligated separately into the vector pBSK(+) and the resulting constructs were used to transform E. coli XL1-Blue cells. Plasmid DNA was prepared from clones containing the assembled inserts and sequenced to confirm the fidelity of the ORFs. The presence of n-1 oligonucleotides (by-products of the oligosynthesis) in the assemblies complicated the build process.
• the synthetic gene was assembled into the vector pBSK(+) between the BamH I and Sal I sites in the polylinker. In this position the gene is not in frame with the alpha peptide and is a significant distance from the lac promoter. However, enough luciferase is produced to enable preliminary characterisation of the enzyme. Crude cell free extracts of E. coli XL1-Blue cells expressing IDRIS (FA) were prepared, from overnight cultures, using the Promega lysis method.
• thermostability of the enzyme in the extract was then tested at 50° C. over 20 minutes and compared with the thermostable mutant E354I+D357Y.
• the new codon optimised triple mutant was significantly more thermostable than the mutant E354I+D357Y ( FIG. 15 ).

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